Project Summary Expression of distinct transcription factors and changes in chromatin states together determine cell- specific gene expression during differentiation of stem and progenitor cells. Defective chromatin and gene dysregulation are integral aspects of tumors such as colorectal cancer, and expression of EZH2 ? a catalytic subunit of Polycomb Repressive Complex (PRC) 2, which places the repressive histone mark H3K27me3 ? is also commonly elevated in colorectal and other cancers. However, the roles and mechanisms of epigenetic modulation in intestinal stem cell (ISC) differentiation and homeostasis remain poorly understood, particularly the relation of H3K27me3 to other epigenetic modifications such as DNA methylation (DNAme). Studies in cancer cell lines or ES cells hint at coordinated gene repression by these alternative processes. However, determinants and reciprocity of the interactions have not been studied critically in vivo, and investigation of repressive chromatin marks lags far behind pre-clinical and clinical development of drugs that affect various epigenetic processes, including small-molecule EZH2 inhibitors. This proposal builds on a general principle we recently uncovered regarding H3K27me3 function in adult tissues and on two additional unexpected observations: Genes massively derepressed in mature PRC2-null intestinal cells are unaffected in ISCs, and H3K27me3-marked genes acquire extensive DNA neomethylation when PRC2 is absent. I propose to study the interdependence of DNAme and PRC2-mediated H327me3 in ISC and mature cell gene expression, differentiation, and tumorigenesis. I will first use engineered mouse models and crypt organoid cultures to eliminate activity of either or both these modifications (Aim1). I will study the consequences using ChIP-seq for the H3K27me3 and other chromatin state modifications, RNA-seq for deep transcriptome analysis, and whole-genome bisulfite sequencing (WGBS) to assess aberrant DNAme. These experiments will elucidate the functional and temporal interactions of H3K27me3 with DNAme for the first time in adult mammalian cells. Secondly, I will extend the questions to mouse models that I have generated for increased or absent EZH2 activity in intestinal tumors (Aim 2). Unexpectedly, I find that EZH2 overexpression reduces, while PRC2 deficiency increases, tumor load significantly. I will characterize tumors that arise in each condition, establish gene expression signatures for tumors with each modulation, and test their dependence on H3K27me3-mediated gene silencing as well as the role of altered DNAme in gene regulation in tumor-derived ISCs. Together, these studies will shed new light on mechanisms of gene silencing in tissue homeostasis and on the functions of specific repressive chromatin modifications in tumors.